1. Field of the Invention
The invention relates to mutants and alleles of the rel gene of coryneform bacteria, which encode variants of GTP-pryophosphate kinase, and to processes for preparing amino acids, in particular L-lysine, L-tryptophan, L-proline, L-valine, L-isoleucine and L-homoserine, by using bacteria which harbor the alleles.
2. Discussion of the Background
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
Amino acids are applied in human medicine, in the pharmaceutical industry, in the food industry and in animal nutrition.
Amino acids are known to be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, continuous efforts are made to improve the production processes. The processes may be improved with respect to fermentation-related measures such as, for example, stirring and oxygen supply or the composition of the nutrient media, such as, for example, sugar concentration during the fermentation, or the working-up into product form, for example by means of ion exchange chromatography, or the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of said microorganisms are improved by applying methods of mutagenesis, selection and mutant choice. This enables strains to be obtained which are resistant to antimetabolites or auxotrophic for metabolites which are of regulatory importance, and produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
For some years now, methods of recombinant DNA technology have likewise been employed in order to improve L-amino acid-producing Corynebacterium strains, by amplifying individual amino acid biosynthesis genes and studying the effect on amino acid production.
The Corynebacterium glutamicum chromosome was sequenced completely some time ago (Kalinowski et al., Journal of Biotechnology 104, 5-25 (2003)). The Corynebacterium efficiens chromosome has likewise been sequenced previously (Nishio et al., Genome Res. 13 (7), 1572-1579 (2003)).
Corresponding sequence information can be found in the public databases. Suitable databases are, for example, the database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), that of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland), the protein Information Resource Database (PIR, Washington, D.C., USA) and the DNA Data Bank of Japan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).
Overviews on the genetics, the metabolism and the technical industrial importance of Corynebacterium can be found in the papers by Ikeda, by Pfefferle et al. and Mueller and Huebner in the book “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering 79, (2003), Springer Verlag, Berlin, Germany, Editor: T. Scheper), in the special edition “A New Era in Corynebacterium glutamicum Biotechnology” of the Journal of Biotechnology (Volume 104 (1-3), 2003, Editor: A. Pühler and T. Tauch), and in the “Handbook of Corynebacterium glutamicum” (Editors: L. Eggeling and M. Bott, CRC Press, Taylor & Francis Group, Boca Raton, Fla., USA, 2005).
The nucleotide sequence of the rel gene coding for GTP-pyrophosphate kinase of Corynebacterium glutamicum is accessible, inter alia in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA) under the access number AF038651. It can furthermore be found as sequence no. 1824 in the patent application EP 1 108 790.
Wehmeier et al. (Microbiology 144, 1853-1862 (1998)) report, inter alia, genetic, microbiological and biochemical studies on a mutant of Corynebacterium glutamicum ATCC 13032 which carries a deletion in the rel gene.
For reasons of better clarity, SEQ ID NO:1 depicts the nucleotide sequence of the rel gene coding for GTP-pyrophosphate kinase of the wild type of Corynebacterium glutamicum (“wild type gene”), according to the information of the NCBI database, and SEQ ID NO:2 or 4 depict the amino acid sequence derived therefrom of the encoded GTP-pyrophosphate kinase. In addition, SEQ ID NO:3 indicates nucleotide sequences located upstream and downstream. The amino acid sequence according to SEQ ID NO: 2 and 4 comprises glycine in position 262. The amino acid sequence of wildtype GTP-pyrophosphate kinase, disclosed in EP 1 108 790 comprises L-glutamic acid in position 262. The nucleotide sequence of the wildtype rel gene according to EP 1 108 790 is depicted in sequence SEQ ID NO:21. SEQ ID NO:22 represents the encoded amino acid sequence.